天津医药

天津医药 ›› 2017, Vol. 45 ›› Issue (12): 1242-1247.doi: 10.11958/20170865

• 实验研究 • 上一篇    下一篇

FOXO3a 调控线粒体自噬在肝脏缺血再灌注 损伤中的作用

宋虎 1， 张建军 2△， 王振 2， 杜晨阳 1， 郑虹 2， 沈中阳 2   

  1. 基金项目： 国家高技术研究发展 863 计划（2012AA021001）； 卫生公益性行业科研专项（201302009）； 天津市器官移植临床医学研究中心 （15ZXLCSY00070） 作者单位： 1 天津医科大学一中心临床学院 （邮编 300192）； 2 天津市第一中心医院器官移植中心 作者简介： 宋虎 （1992）， 男， 硕士研究生， 主要从事细胞自噬在肝脏缺血再灌注损伤以及肝癌中的作用等相关研究 △通讯作者 E-mail：zhangjianjun99@medmail.com.cn
  • 收稿日期:2017-08-04 修回日期:2017-10-11 出版日期:2017-12-15 发布日期:2017-12-15
  • 通讯作者: 张建军 E-mail:zhangjianjun99@medmail.com.cn
  • 基金资助:
    国家高技术研究发展计划（863）;卫生公益性行业科研专项

Effects of FOXO3a on regulating mitophagy in hepatic ischemia reperfusion injury

SONG Hu1, ZHANG Jian-jun2△, WANG Zhen2, DU Chen-yang1, ZHENG Hong2, SHEN Zhong-yang2   

  1. 1 First Central Clinical College of Tianjin Medical University, Tianjin 300192, China; 2 Organ Transplant Center, Tianjin First Center Hospital △Corresponding Author E-mail: zhangjianjun99@medmail.com.cn
  • Received:2017-08-04 Revised:2017-10-11 Published:2017-12-15 Online:2017-12-15

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摘要： 目的 探讨转录因子 FOXO3a 调控线粒体自噬对小鼠肝脏缺血再灌注损伤的影响。方法 雄性 C57BL/6 小鼠 30 只， 随机分为 Sham 组 （只模拟开腹手术不夹闭） 和再灌注 （IR） 2、 6、 12、 24 h 组， 每组 6 只， 建立小鼠肝缺血再 灌注损伤模型。血生化检测各组丙氨酸转氨酶（ALT）、 天冬氨酸转氨酶（AST）变化， HE 染色及 TUNEL 法观察肝组 织损伤及细胞凋亡情况， Western blot 及 qRT-PCR 检测各组细胞中转录因子 FOXO3a、 线粒体自噬相关蛋白 Nix 蛋 白及其 mRNA 表达水平。培养小鼠肝 AML12 细胞， 用 FOXO3a 和 Nix 干扰 RNA 处理细胞建立缺氧 1.5 h 复氧 6 h 的模型， 分为 siRNA-NC 组（加入 10 μL siRNA 空载对照转染细胞）、 FOXO3a siRNA 组（加入 10 μL FOXO3a siRNA 转染细胞）以及 Nix siRNA 组（加入 10 μL Nix siRNA 转染细胞）。MTT 法检测各组细胞活力， 共聚焦显微镜观察各 组细胞内自噬体的数量与分布， Western blot 检测 FOXO3a、 Nix、 微管相关蛋白 LC3、 凋亡蛋白 P62 及 Caspase-3 的表 达情况。结果 IR 各组 ALT、 AST 均明显升高， 且再灌注 6 h 时达到峰值（P＜0.05）。HE 及 TUNEL 结果示再灌注 6 h 时小鼠肝组织损伤以及细胞凋亡最严重。IR 各组 FOXO3a 及 Nix 的 mRNA 相对表达量均高于 Sham 组， 且再灌 注 12 h 时 FOXO3a 的 mRNA 表达量最高， 再灌注 6 h 时 Nix 的 mRNA 表达量最高（P＜0.05）。Western blot 示再灌 注 12 h 时 FOXO3a 相对表达水平最高， 再灌注 6 h 时 Nix、 Caspase-3、 LC3Ⅱ相对表达水平最高。干扰 FOXO3a 的表 达后， MTT 显示 FOXO3a siRNA 组细胞存活率降低（P＜0.05）； Western blot 检测显示 siRNA-NC 组 FOXO3a 表达水 平高于 FOXO3a siRNA 组， Nix、 Caspase-3、 LC3Ⅱ表达水平明显低于 FOXO3a siRNA 组 （P＜0.05）。共聚焦显微镜观 察示 siRNA-NC 组细胞内自噬体的数量低于 FOXO3a siRNA 组。干扰 Nix 的表达后， MTT 显示 Nix siRNA 组细胞存 活率升高 （P＜0.05）； Western blot 检测显示 siRNA-NC 组 Nix、 P62、 LC3Ⅱ表达水平高于 Nix siRNA 组， 而 FOXO3a 表 达水平低于 Nix siRNA 组 （P＜0.05）。结论 FOXO3a 可减轻小鼠肝缺血再灌注损伤， 其机制可能与 FOXO3a 抑制肝 细胞线粒体自噬且抑制细胞凋亡有关。

关键词: FOXO3a, 线粒体自噬, 缺血再灌注损伤, 凋亡

Abstract: Objective To investigate the effect of transcription factor FOXO3a on mitophagy in hepatic ischemiareperfusion injury in mice. Methods A total of 30 male C57BL/6 mice were randomly divided into Sham operation group (Sham) and ischemia reperfusion (IR) 2 h, 6 h, 12 h and 24 h groups, 6 mice in each group. The mouse model of liver ischemia -reperfusion injury was established. Blood biochemical methods were used to detect changes of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). HE staining and TUNEL were used to observe the damages of liver tissue and apoptosis. Western blot assay and qRT-PCR were used to detect the expressions of transcription factor FOXO3a, mitochondrial autophagy-related protein Nix protein and its mRNA expression in each group. Mouse liver AML12 cells were treated with FOXO3a and Nix interfering RNA, and the model was established for 6 h after hypoxia for 1.5 h.These cells were divided into siRNA-NC group, FOXO3a siRNA group and Nix siRNA group. MTT assay was used to detect the viability of cells in each group. The number and distribution of autophagy in each group were observed by confocal microscopy. The expressions of FOXO3a, Nix, microtubule-associated protein LC3, apoptotic protein P62 and Caspase-3 were detected by Western blot assay. Results The levels of ALT and AST in all groups of IR were reduced, and reached the peak value at 6 h (P＜0.05). HE and TUNEL results showed that liver injury and apoptosis were the most serious at 6 h after reperfusion. The expression of FOXO3a and Nix was higher in IR group than that in the Sham group, and the expression level of FOXO3a mRNA was the highest at 12 h after reperfusion, the expression of Nix mRNA was the highest at 6 h after reperfusion (P＜0.05). Western blot assay showed the highest expression of FOXO3a in the reperfusion of 12 h, and the highest expression levels of Nix, Caspase-3 and LC3Ⅱin reperfusion 6 h. After interfering with the expression of FOXO3a, MTT showed a marked reduction in cell survival (P＜0.05), Western blot assay showed that the expression level of FOXO3a was significantly higher in siRNA-NC group than that in FOXO3a siRNA group, and the expression levels of Nix, Caspase-3 and LC3Ⅱwere significantly lower than those of FOXO3a siRNA group. Confocal microscopy showed that the number and distribution of autophagosomes were significantly lower in siRNA-NC group than those in FOXO3a siRNA group. After interfering with the expression of Nix, MTT showed a marked increase in cell survival (P＜0.05), Western blot assay showed that the expression levels of Nix, P62 and LC3Ⅱ were significantly higher in siRNA-NC group than those in Nix siRNA group. Conclusion FOXO3a can reduce the hepatic ischemia-reperfusion injury in mice, which may be related to the FOXO3a inhibition for liver cell mitophagy and apoptosis.

Key words: FOXO3a, mitophagy, ischemia-reperfusion injury, apoptosis